Acquired immunodeficiency syndrome (AIDS) in people living with human immunodeficiency virus (PLWH) is associated with an increased risk for a number of different malignances. The most common cancers within this population are Kaposi's sarcoma (KS) and non-Hodgkin's lymphomas, such as primary effusion lymphoma (PEL). While KS is an endothelial cell-derived lesion PEL is of B cell origin. The etiological agent of KS and PEL is the oncogenic DNA virus, Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV displays two distinct phases of its viral lifecycle, latency and lytic infection, and both are required for the progression to and maintenance of KSHV-associated cancers. The remodeling of cellular gene expression during infection plays an essential role in the establishment of latency as well as progression to the lytic phase. Thus, deciphering the gene regulatory mechanisms operating during KSHV infection will identify processes involved in tumorigenesis in PLWH as well as identify potential targets for therapeutic development. Transposable elements (TEs) are genetic sequences that can, or at one time could, move around the genome from one location to another and recent analyses of the genetic regulatory landscape of mammalian cells have revealed TEs function in a wide range of regulatory processes, including transcriptional regulation. While the function of TE regulatory elements has gained appreciation in areas such as stem cell biology, the role of TEs in infectious diseases and cancer, such as in PLWH is not known. Our study investigates this unknown which is the basis of provocative question 2 (PQ#2). Leveraging 5'-end RNA sequencing technology we have mapped the transcription initiation landscape of latent and lytic PEL cells and have identified wide-spread transcription of TEs. Moreover, we discovered TEs transcribed from within annotated gene regulatory elements such as enhancers and gene promoters, and that some transcribed TEs are bound by the main KSHV-encoded transcription factor, RTA. Remarkably, a targeted CRISPR screen identified an RTA-bound TE-derived enhancer that significantly affects KSHV lytic reactivation in PEL. Together with observations from the scientific literature our unpublished data form the scientific premise for the proposed studies. Our central hypothesis is that TE-derived gene regulatory elements are at the center of the host-KSHV battle and that their modulation is essential for pathogenesis. To test this hypothesis, we propose an integrated series of experiments aimed at determining the interplay between TEs and KSHV. In Aim 1, we will determine the function of RTA-bound TEs in PEL. In Aim 2, we will determine how KSHV infection of endothelial cells influences TE expression and the consequences of TE-derived regulatory elements on the viral lifecycle as well as cellular pathways required for tumorigenesis. Completion of these studies is expected to determine how TEs contribute to KSHV-associated cancer in PLWH...